The goal of the PI is to elucidate the mechanisms of the modulatory action of TGF-beta on PTEN expression that is critical to understand the pathogenesis of malignant pancreatic tumors. Our preliminary data show that TGF-beta functionally affects activation, stability, and transcription of PTEN via SMAD-dependent and - independent pathways. Our overall hypothesis is that TGF-beta modulates PTEN expression in pancreatic cancer, and this modulation converts TGF-beta from a tumor suppressor to a tumor promoter. Our Specific Aims center on this hypothesis, which are designed to determine the mechansims for the dual functional roles of TGF-beta. Our first aim studies how K-RAS modulates TGF-beta-induced, SMAD-dependent PTEN expression, as K-RAS is activated in >90% of pancreatic cancers. Preliminary data indicate that inhibition of oncogenic K-RAS improves SMAD nuclear translocation and reverses TGF-beta-induced PTEN suppression. We suggest that this PTEN upregulation is key to SMAD-dependent signaling being tumor suppressive. Our second aim studies TGF-beta-induced regulation of PTEN at post-translational levels. Based on our data, TGF-beta has at least two effects on reducing PTEN in pancreatic cancer - suppression of PTEN transcription and reduction of the cytoplasmic PTEN pool. The PI will determine if casein kinase regulates PTEN stability, and whether C-terminus phosphorylation of PTEN can be affected by TGF-beta- induced downregulation of casein kinase. Our third aim examines TGF-beta-induced PTEN expression that is SMAD-independent, and particularly focuses on NF-kB as the mediator. TGF-beta-induced PTEN suppression in our pancreatic cell models occurred in (a) SMAD4-null cells, and (b) with oncogenic K-RAS, essentially completely inhibiting SMAD-dependent signaling. We infer from our data that NF-kB is an excellent candidate for this SMAD-independent pathway. The strategy for these studies will combine pharmacological, molecular, and biochemical approaches to characterize pathways that regulate PTEN expression in pancreatic cancer. These studies should provide novel insights into the precise mechanisms that regulate the modulatory effect of the TGF-beta on PTEN expression. The research proposed here will explore basic understanding and treatment to patients with pancreatic cancer, and serve as an outstanding mentored project for the Pi's cancer research development.